A variety of blood diseases are caused by mutations involving the structure or expression of erythroid proteins. Mutations involving non-globin erythroid genes are associated with a multitude of disorders, including porphyria, sideroblastic anemia, and glucose-6-phosphate dehydrogenase deficiency. Genetic aberrations in globin gene expression result in several common blood diseases, including sickle cell anemia and .beta.-thalassemia.
Sickle cell anemia is an autosomal recessive disorder involving a mutation in the .beta.-globin gene that causes hemoglobin to form long polymers under deoxygenated conditions. As a result, the red blood cell is deformed and assumes a "sickle" shape which may compromise the micro-circulation. Patients with this disorder have chronic anemia and typically suffer painful "sickle cell crises" and multiple end-organ damage from obstruction of blood vessels with sickled red blood cells. Medical therapy for sickle cell anemia has been largely directed toward managing the complications of vascular insufficiency caused by red cell deformation, although allogeneic bone marrow transplantation, which supplies normal red blood cells, has been shown to be effective (Johnson, et al., N. Eng. J. Med. 311:780-783, 1984).
Thalassemias are disorders associated with a diminished rate of globin synthesis, which may be a consequence of a deletion of the globin gene itself, or, more commonly, are due to mutations in regulatory sequence information. .beta.-thalassemia is one of the most frequent single gene disorders in humans; 50,000 children are born yearly with this disease. It is marked by anemia, failure to thrive, and splenomegaly. Iron deposition caused by increased absorption and multiple transfusions often results in multiple organ system failure. As with sickle cell anemia, allogeneic bone marrow transplantation has been shown to be curative of thalassemia (Thomas et al., Lancet 2:227-228, 1982).
However, the use of allogeneic bone marrow transplant to treat either sickle cell anemia or .beta.-thalassemia is problematic. First, the availability of a suitable donor is frequently limited. Further, in order to avoid the complication of graft vs. host disease, immunosuppressive drugs are typically administered, which are themselves associated with increased risk of infection, cancer, and a substantial mortality rate.
A potential alternative to replacement of defective red blood cells in these conditions is correction of the underlying genetic defect by introducing a normal copy of the erythroid gene that is missing or defective into the erythroid cells of the patient. The ability to design safe and efficient delivery systems for gene replacement therapy is key to improving the prospects for therapeutic intervention.
Gene delivery has been accomplished using viruses engineered to carry foreign genetic material into a cell of interest. Such viruses have included DNA viruses such as vaccinia, adenovirus, and adeno-associated virus (AAV) and RNA viruses such as retroviruses.
Retroviruses, which naturally infect and integrate their genome into a recipient host cell, are ideal vehicles for gene transfer. In order to maximize safety, the recombinant viral genome may be introduced and encapsulated in a packaging cell line to generate a replication-incompetent retroviral stock. Such virus is able to infect a target cell and introduce the gene of interest, but cannot replicate. This feature limits the function of the viral vector to being a gene transfer vehicle, and precludes the generation of potentially dangerous infectious retroviruses.
Viruses have been used to transfer genes into erythroid cells. For example, recombinant AAV vector was shown to transduce a human .gamma.-globin gene into human erythroid K562 cells (Walsh et al., Proc. Natl. Acad. Sci. 89:7257-7261, 1992). Moreover, initial experiments, engineering retroviruses to carry globin genes using the cis-acting regulatory elements linked to the .beta.-globin gene, resulted in low .beta.-globin expression in erythroid cells and hematopoietic stem cells (Cone et al., Mol. Cel. Biol. 7:887-897, 1987; Karlsson et al., Proc. Natl. Acad. Sci. 84:2411-2415, 1987; Miller et al., J. Virol. 62:4337-4345, 1988; Dzierzak et al., Nature 331:35-41, 1988).
Subsequently, a major regulatory region far upstream of the .beta.-globin gene, the .beta.-locus control region (.beta.-LCR) was identified (Grosveld et al., Cell 51:975, 1987). Retroviruses containing the .beta.-globin gene under .beta.-LCR control were found to express higher levels of .beta.-globin (Novak et al., Proc. Natl. Acad. Sci. 87:3386-3390, 1990; Chang et al., Proc. Natl. Acad. Sci. 89:3107-3110, 1992; Plavec et al., Blood 81:1384-1392, 1993). Unfortunately, while expression of the .beta.-globin gene was initially improved, the .beta.-LCR element was highly recombinogenic, and frequent rearrangement of the viral sequences occurred before or after integration into host cells. This instability resulted in the production of low-titer retroviral stocks that infected hematopoietic stem cells with low efficiency.
A major upstream regulatory region of the .alpha.-globin gene cluster, the .alpha.-locus control region (.alpha.-LCR), was identified more recently and shown to function in an enhancer-like manner to increase gene expression (Higgs et al., Genes and Dev. 4:1588-1601, 1990; Ren et al., Blood 81: 1058-1066, 1993). The region is located 40 kb upstream of the gene cluster and its function is erythroid-specific. .alpha.LCR was localized to a 255 bp element (SEQ ID NO.6) and shown to confer inducible expression on a heterologous promoter (Jarman et al., Mol. Cell Biol. 11:4679-4689, 1991; Pondel et al., Nucleic Acids Res. 20:237-243, 1992; Ren et al., Blood 81:1058-1066, 1993).